Exploring New Virulence Factors of the Oral Spirochete Treponema denticola

描述(由申请人提供)：齿密螺旋体(TD)与严重和难治性牙周疾病密切相关。作为红色复合体细菌的一员，TD主要生活在与牙菌斑直接接触的牙菌斑的根尖表面 上皮组织。因此，TD处于直接遭遇巨大宿主免疫攻击的前沿，例如补体杀伤和吞噬作用。在口袋里，中性粒细胞(PMN)是主要的免疫细胞，通过吞噬杀死病原体来保护宿主。此外，牙周沟液(GCF)主要由血清组成，含有补体系统，这是宿主防御的关键成分。以前的报道表明，TD对补体杀伤具有抵抗力，能够调节中性粒细胞，并能在口腔菌群中茁壮成长。然而，其中涉及的分子机制仍然难以捉摸。这一应用的中心假设是TD的一个新的毒力因子TDE0362(TMAC)具有双重功能：它的C端(C362)是一种蛋白酶，它能切割对PMN的激活和补体杀伤至关重要的免疫因子；它的N端(N362)包含一个细菌免疫球蛋白(Big)样结构域，通过分子模仿机制阻止PMN的激活。总的来说，TMAC保护TD免受吞噬和补体杀伤，从而提高其存活率和感染的建立。为了验证这一假说，这一应用将集中于以下特定目的：(1)确定TMAC是否被切割和分泌，以及参与切割的机制；(2)研究TMAC对人免疫球蛋白G的内肽酶活性；(3)阐明TMAC在PMN激活中的作用及其涉及的机制；(4)探讨TMAC在TD体外和体内致病中的作用。该项目的完成将促进我们对TD致病机制的了解，特别是对先天性免疫逃避机制的了解，这可能会导致针对牙周病的新的治疗干预措施。

DESCRIPTION (provided by applicant): The spirochete Treponema denticola (Td) is strongly associated with severe and refractory periodontal conditions. As a member of the 'red-complex' bacteria, Td primarily lives on the apical surface of subgingival plaque in direct contact with the epithelium. Thus Td is at the forefront that directly encounters enormous host immune attacks, e.g., complement killing and phagocytosis. In the pocket, polymorphoneclear leukocytes (PMNs) are the major immune cells that protect the host by killing pathogens via phagocytosis. In addition, the gingival crevice fluid (GCF), which is mainly composed of serum, contains the complement system, a critical component in host defense. Previous reports suggest that Td is resistant to the complement killing, and is able to modulate PMNs and can thrive in the oral flora. However, the molecular mechanisms involved remain elusive. The central hypothesis of this application is that TDE0362 (Tmac), a new virulence factor of Td, has dual functions: its C-terminus (C362) is a protease that cleaves immune factors that are essential for the activation of PMNs and the complement killing; its N-terminus (N362) contains a bacterial immunoglobulin (Big)-like domain which blocks PMN activation via a molecular mimicry mechanism. Collectively, Tmac protects Td from the phagocytosis and the complement killing, and consequently enhances its survival and establishment of infection. To test this hypothesis, this application wil focus on the following Specific Aims: (1) To determine if Tmac is cleaved and secreted, and the mechanism involved in the cleavage; (2) To study the endopeptidase activity of Tmac on human IgG; (3) To elucidate the role of Tmac in PMN activation and its mechanism involved; and (4) To investigate the role of Tmac in the pathogenicity of Td in vitro and in vivo. Completion of this project will advance our current understanding of the pathogenicity of Td, in particular, the mechanisms involved in the innate immune evasion, which could potentially lead to new therapeutic interventions against periodontal diseases.